.alpha.-Amylase enzymes have been used industrially for a number of years and for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing and baking. A further use of .alpha.-amylases which is becoming increasingly important is the removal of starchy stains during washing or dishwashing.
In recent years attempts have been made to construct .alpha.-amylase variants having improved properties with respect to specific uses such as starch liquefaction and textile desizing.
For instance, U.S. Pat. No. 5,093,257 discloses chimeric .alpha.-amylases comprising an N-terminal part of a B. stearothermophilus .alpha.-amylase and a C-terminal part of a B. lichenifonnis .alpha.-amylase. The chimeric .alpha.-amylases are stated to have unique properties, such as a different thermostability, as compared to their parent .alpha.-amylase. However, all of the specifically described chimeric .alpha.-amylases were shown to have a decreased enzymatic activity as compared to their parent .alpha.-amylases.
EP 252 666 describes hybrid amylases of the general formula Q-R-L, in which Q is a N-terminal polypeptide residue of from 55 to 60 amino acid residues which is at least 75% homologous to the 57 N-terminal amino acid residues of a specified .alpha.-amylase from B. amyloliquefaciens, R is a specified polypeptide, and L is a C-terminal polypeptide comprising from 390 to 400 amino acid residues which is at least 75% homologous to the 395 C-terminal amino acid residues of a specified B. licheniformis .alpha.-amylase.
Suzuki et al. (1989) disclose chimeric .alpha.-amylases, in which specified regions of a B. amyloliquefaciens .alpha.-amylase have been substituted for the corresponding regions of a B. licheniformis .alpha.-amylase. The chimeric .alpha.-amylases were constructed with the purpose of identifying regions responsible for thermostability. Such regions were found to include amino acid residues 177-186 and amino acid residues 255-270 of the B. amyloliquefaciens .alpha.-amylase. The alterations of amino acid residues in the chimeric .alpha.-amylases did not seem to affect properties of the enzymes other than their thermostability.
WO 91/00353 discloses .alpha.-amylase mutants which differ from their parent .alpha.-amylase in at least one amino acid residue. The .alpha.-amylase mutants disclosed in said patent application are stated to exhibit improved properties for application in the degradation of starch and/or textile desizing due to their amino acid substitutions. Some of the mutants exhibit improved stability, but no improvements in enzymatic activity were reported or indicated. The only mutants exemplified are prepared from a parent B. licheniformis .alpha.-amylase and carry one of the following mutations: H133Y or H133Y+T149I. Another suggested mutation is A111T.
FR 2,676,456 discloses mutants of the B. licheniformis .alpha.-amylase, in which an amino acid residue in the proximity of His 133 and/or an amino acid residue in the proximity of Ala 209 have been replaced by a more hydrophobic amino acid residue. The resulting .alpha.-amylase mutants are stated to have an improved thermostability and to be useful in the textile, paper, brewing and starch liquefaction industry.
EP 285 123 discloses a method of performing random mutagenesis of a nucleotide sequence. As an example of such sequence a nucleotide sequence encoding a B. stearothermophilus .alpha.-amylase is mentioned. When mutated, an .alpha.-amylase variant having improved activity at low pH values is obtained.
In none of the above references is it mentioned or even suggested that .alpha.-amylase mutants may be constructed which have improved properties with respect to the detergent industry.
EP525 610 relates to mutant enzymes having improved stability towards ionic tensides (surfactants). The mutant enzymes have been produced by replacing an amino acid residue in the surface part of the parent enzyme with another amino acid residue. The only mutant enzyme specifically described in EP 525 610 is a protease. Amylase is mentioned as an example of an enzyme which may obtain an improved stability towards ionic tensides, but the type of amylase, its origin or specific mutations are not specified.
WO 94/02597 discloses .alpha.-amylase mutants which exhibit improved stability and activity in the presence of oxidizing agents. In the mutant .alpha.-amylases, one or more methionine residues have been replaced with amino acid residues different from Cys and Met. The .alpha.-amylase mutants are stated to be useful as detergent and/or dishwashing additives as well as for textile desizing.
WO 94/18314 discloses oxidatively stable .alpha.-amylase mutants, including mutations in the M197 position of B. licheniformis .alpha.-amylase.
EP 368 341 describes the use of pullulanase and other amylolytic enzymes optionally in combination with an .alpha.-amylase for washing and dishwashing.
An object of the present invention is to provide .alpha.-amylase variants which--relative to their parent .alpha.-amylase--possess improved properties of importance, inter alia, in relation to the washing and/or dishwashing performance of the variants in question, e.g. increased thermal stability, increased stability towards oxidation, reduced dependency on Ca.sup.2+ ion and/or improved stability or activity in the pH region of relevance in, e.g., laundry washing or dishwashing. Such variant .alpha.-amylases have the advantage, among others, that they may be employed in a lower dosage than their parent .alpha.-amylase. Furthermore, the .alpha.-amylase variants may be able to remove starchy stains which cannot, or can only with difficulty, be removed by .alpha.-amylase detergent enzymes known today.